For the next 3 year period we propose to continue our work on P-450 hemeproteins which demonstrated the existence of a well defined domain designed for binding of heme and substrate, in at least 5 different monoxygenases: P-450CAM, P-450LM-2, P-450LM-4, P-450SCC and P-45011 beta. The location of substrate and inhibitor binding sites within this domain was detained by photocovalent attachment of suitable arylazido labels. These results together with RIAs on the proteins and their hemepeptides strongly support our hypothesis that P-450s exhibit a common molecular design. It is our long-range goal to unravel this design by continued sequence, photoaffinity labeling and other specific chemical experiments to furnish the intimate details required for a better understanding of the mechanism of action of P-450 hemeproteins. Associated objectives are identification of the salient molecular differences between mitochondrial-bacterial and microsomal P-450s and the function-related structural features which distinguish P-450LM-2 and P-450LM-4 while generating striking resemblance between P-450LM-2 rabbit liver and P-450CAM. We propose to establish the position within the linear polypeptide chain of the heme-liganding cysteine which may be used as a reference point for sequence comparisons. We address this problem in the context of specific chemical derivatization and sequence work and emphasize that substrates may also be bound by special cysteine residues which can be pinpointed by this approach. This will afford valuable overlaps for sequence assignments and furnish important information on the molecular architecture of P-450 hemoproteins. It is proposed to isolate P-450s from camphor-treated rat liver microsomes by affinity chromatography, to enhance the purity of adrenocortical microsomal P-450s and reductase, and to isolate hemepeptides by a new photoaffinity chromatography procedure.